PROJECT SUMMARY/ABSTRACT Mycobacterium tuberculosis (Mtb) infects one-third of the human population and causes ~1.7 million deaths a year, broadly impacting population and economic development. Current therapy requires a prolonged time-course of 6-9 months, making patient compliance difficult, and exacerbating the problem of drug-resistant strains. A critical factor in the prolonged therapy required is thought to be heterogeneity in microenvironments and bacterial and lesion properties during infection, but little is known about what drives this heterogeneity, and how it affects colonization and disease progression. Our long-term hypothesis is that the heterogeneous lesions, microenvironments and bacterial responses are regulated and not just stochastic, providing points for therapeutic intervention. The inability to understand this process thus constitutes a critical block that must be overcome for continued progress in the field. Bulk assays do not provide a means to study this phenomenon, and the technical difficulties associated with studying heterogeneity during whole animal infection has been a significant barrier to progress. This project seeks to overcome these hurdles by first developing an imaging strategy to enable analysis of Mtb-host interactions at the single bacterium level, in the context of intact 3- dimensional host tissue architecture. To accomplish this, tissue optical clearing methods and innovative fluorescent reporter Mtb strains that allow direct readout of a bacterium?s replication status and aspects of its local environment will be used, together with a murine infection model that exhibits the full range of granuloma types observed during human infection. This integrated imaging strategy will then be utilized to (i) establish a framework delineating key lesion and host cell properties conducive for Mtb growth, and (ii) elucidate the impact of non-uniform microenvironments on Mtb replication and lesion properties in vivo, and test how modulation of Mtb response to environmental signals impacts infection heterogeneity and outcome. The latter aim will focus on the response of Mtb to chloride and potassium, two novel and important cues for Mtb during infection that represent possible Achilles? heels that can be targeted to shift the balance of infection. Mechanistic understanding of heterogeneity in microenvironments and lesion properties is critical for defining the molecular and cellular basis of how Mtb interfaces with its host, vital for the development of better therapies. Further, the development of a framework for understanding Mtb-host interactions represents a rich source for hypothesis generation for the long-term goal of understanding the host and bacterial determinants that influence heterogeneity, and how these elements control Mtb colonization, disease progression, and treatment. The concept of heterogeneous environments and its impact on host-pathogen interactions is increasingly being appreciated, with studies in multiple bacterial species. The methodological and conceptual advances achieved from the proposed studies will thus also have far-reaching applicability to the broader field of infectious diseases and microbial pathogenesis.